Reducing the proliferation of adenocarcinoma cells (CaCo-2) by administration of a poly-oxygenated metal hydroxide

ABSTRACT

A method of treating a mammal, comprising administering a therapeutically effective amount of a poly-oxygenated metal hydroxide composition to a mammal to reduce a proliferation of hypoxic carcinoma cells, wherein the poly-oxygenated metal hydroxide composition comprises a clathrate containing free oxygen gas (O 2 ) molecules. The carcinoma cells may comprise colon adenocarcinoma cells (CaCo-2). The poly-oxygenated metal hydroxide material is configured to provide bioavailable oxygen gas molecules to a mammal when administered to the mammal. The poly-oxygenated metal hydroxide composition can be administered intravenously, directly to carcinoma cells, and orally. The composition may comprise a fluid, where the poly-oxygenated metal hydroxide composition is soluble in the fluid.

CLAIM OF PRIORITY

This application is a Continuation-in-Part (CIP) of U.S. patentapplication U.S. Ser. No. 15/797,972 filed Oct. 30, 2017, entitledREDUCING THE PROLIFERATION OF CARCINOMA CELLS BY ADMINISTRATION OF APOLY-OXYGENATED METAL HYDROXIDE, which is a Continuation-in-Part (CIP)of U.S. patent application U.S. Ser. No. 15/183,403 filed Jun. 15, 2016,entitled INTRAVENOUS ADMINISTRATION OF AN OXYGEN-ENABLE FLUID, whichclaims priority of U.S. Provisional Patent Application U.S. Ser. No.62/315,524 entitled OXYGEN-ENABLED RESUSCITATIVE FLUID filed Mar. 30,2016, the teachings of which are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

This disclosure is directed to a method of delivering an oxygen-enabledsolution to a mammal to reduce the proliferation of carcinoma.

BACKGROUND

When blood is lost, the chief immediate need is to cease further bloodloss followed by replacing the lost blood volume. This critical need isimportant for allowing the remaining red blood cells to oxygenate bodytissue albeit at a reduced capacity. When the body detects the lowerhemoglobin levels, from extreme blood loss, compensatory mechanismsbegin. There are currently no resuscitative fluids that provide oxygento hypoxic cells and tissues following major blood loss.

Oxygen therapeutics have traditionally been categorized as eitherhemoglobin-based oxygen carriers (HBOCs) or perfluorocarbons (PFCs).Unlike blood, HBOCs and PFCs do not require blood typing, have a longshelf life, do not transmit blood borne diseases, and in most cases donot need refrigeration. Despite these promising attributes thewide-spread utility of HBOCs and PFCs has been limited by concernsregarding hypertension from systemic arteriolar constriction andleukocyte activation, respectively.

Mammals with carcinoma suffer from the proliferation of carcinoma cells.Many such carcinoma cells are hypoxic.

SUMMARY

A method of treating a mammal, comprising administering atherapeutically effective amount of a poly-oxygenated metal hydroxidecomposition to a mammal to reduce a proliferation of hypoxic carcinomacells, wherein the poly-oxygenated metal hydroxide composition comprisesa clathrate containing free oxygen gas (O₂) molecules. The carcinomacells may comprise colon adenocarcinoma cells (CaCo-2). Thepoly-oxygenated metal hydroxide material is configured to providebioavailable oxygen molecules to a mammal when administered to themammal. The poly-oxygenated metal hydroxide composition can beadministered intravenously, directly to carcinoma cells, and orally. Thecomposition may comprise a fluid, where the poly-oxygenated metalhydroxide composition is soluble in the fluid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method of intravenously administering a mammal atherapeutically effective amount of a poly-oxygenated metal hydroxide inaccordance with this disclosure;

FIGS. 2A-2D are diagrams illustrating systemic characteristics of 50%isovolemic hemodilution, including hematocrit, heart rate, mean arterialpressure, and pulse pressure. Measurements were taken immediately priorto (BL) and following (HD t0) hemodilution;

FIG. 3A shows tissue oxygenation (P_(ISF) O₂) following 50% volumereplacement using Ox66™ in a crystalloid. All P_(ISF) O₂ values (mmHg)were normalized to baseline (BL) for ease of comparison;

FIG. 3B shows tissue oxygenation (P_(ISF) O₂) following 50% volumereplacement using Ox66™ in a crystalloid, using particles smaller thanthose in FIG. 3B, and further shows tissue oxygenation using PEGylatedOx66™ particles in a Colloid;

FIG. 3C shows survival results of specimens after undergoing hemorrhagicshock following resuscitation using PEGylated Ox66™ particles in aColloid, including complete survival of one specimen;

FIGS. 4A and 4B show systemic parameters including heart rate and meanarterial pressure following isovolemic hemodilution with test solutions;

FIG. 5 shows arteriolar luminal diameters. Arterioles included weresmaller than 60 microns at baseline;

FIG. 6 shows the proliferation of hepatocarcinoma cells (HEPG-2)significantly reduced following administration with variousconcentrations of Ox66™;

FIG. 7A and FIG. 7B illustrate images of cells HEPG-2 cells prior todosing and after dosing, respectively;

FIG. 8 shows the proliferation of prostrate carcinoma cells (22Rv1)significantly reduced following administration with variousconcentrations of Ox66™;

FIG. 9 shows the proliferation of lung carcinoma cells (A549)significantly reduced following administration with variousconcentrations of Ox66™; and

FIG. 10 shows the proliferation of colon adenocarcinoma cells (CaCo-2)significantly reduced following administration with variousconcentrations of Ox66™.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The following description of exemplary embodiments provides informationthat enables a person skilled in the art to make and use the subjectmatter set forth in the appended claims, but may omit certain detailsalready well-known in the art. The following detailed description is,therefore, to be taken as illustrative and not limiting.

The example embodiments may also be described herein with reference tospatial relationships between various elements or to the spatialorientation of various elements depicted in the attached drawings. Ingeneral, such relationships or orientation assume a frame of referenceconsistent with or relative to a patient in a position to receivetreatment. However, as should be recognized by those skilled in the art,this frame of reference is merely a descriptive expedient rather than astrict prescription.

Despite what is known from physiological principles, there is nopractice-based evidence to suggest colloid solutions offer substantiveadvantages over crystalloid solutions with respect to hemodynamiceffects. In addition, there is no evidence to recommend the use of othersemisynthetic colloid solutions. Balanced salt solutions are reasonableinitial resuscitation fluids, although there is limited practice-basedevidence regarding their safety and efficacy. Additionally, the safetyof hypertonic solutions has not been established. Ultimately, theselection of the specific resuscitative fluid should be based onindications, contraindications, and potential toxic effects in order tomaximize efficacy and minimize toxicity. In addition, the capability ofa resuscitative fluid to carry oxygen, as well as to maximize efficacyand minimize toxicity, is desperately needed.

According to this disclosure, there is a significant therapeutic benefitto intravenously oxygenate blood of a human individual and animal,collectively mammals, and create a more effective resuscitative fluidusing a poly-oxygenated metal hydroxide, and particularly nano-sizedpoly-oxygenated aluminum hydroxide, such as Ox66™ oxygen carryingparticles manufactured by Hemotek, LLC of Plano, Tex. Ox66™ is an oxygencarrying powder that contains about 66% oxygen, and includes a trueclathrate that is a lattice-like structure that provides large areascapable of capturing and holding O_(2(g)) oxygen gas. The Ox66™poly-oxygenated aluminum hydroxide has a molecular formula Al₁₂H₄₂O₃₆,and the O_(2(g)) oxygen gas molecules are bioavailable to, and used bythe body, because the O_(2(g)) oxygen gas molecules are not bound in thehydroxide complex. Ox66™ exists under STP (standard temperature andpressure) as a poly-oxygenated aluminum hydroxide comprising aclathrate, and chlorine. The molecular formula Al₁₂H₄₂O₃₆ ismathematically reduced to the molecular formula Al(OH)₃.6O₂. The 6 freeoxygen gas molecules (O_(2(g))) are separate from the oxygen moleculescovalently bound in the hydroxide complex. The hydrogen is effervescent.The poly-oxygenated aluminum hydroxide is soluble in a fluid.

This disclosure significantly increases tissue oxygenation of themammal, known as oxygen tension PO₂. In certain applications of Ox66™,the PO₂ levels of a hemo-diluted mammal can exceed baseline. Fluidresuscitation with colloid and crystalloid solutions is a globalintervention in acute medicine, and while the selection and ultimate useof resuscitation fluids is based on physiological principles, clinicianpreference determines clinical use. Studies have shown that Ox66™ doesnot create any negative effects in toxicology studies where Ox66™ waseither injected or gavaged in a mammal.

With enough blood loss, like in amputations and other military traumasituations, red blood cell levels drop too low for adequate PO₂ tissueoxygenation, even if volume expanders maintain circulatory volume theydo not deliver oxygen. In these situations, the only currently availablealternatives are blood transfusions, packed red blood cells, or a noveloxygen-enabled resuscitative fluid according to this disclosure.

This disclosure provides a novel oxygen-enabled blood additive, alsoreferred to as a resuscitative fluid, that can effectively oxygenatemammal tissues and provide essential elements to protect and savecritical cells and tissues, and the mammal itself. This disclosure isdesperately needed on the battlefield, as well as in civilian traumacases. One exemplary formulation consists of a fluid of 75-90% colloidor crystalline solutions with 10-25% addition of a poly-oxygenated metalhydroxide material, such as but not limited to, nano-sized Ox66™particles, resulting in concentration ranges of 0.1 mg/l to 1000 mg/l.For use as a blood additive, ideal sizes of the Ox66™ particles may bebetween 10 nm to 100 μm in size, depending on the treatment. To avoidimmune response, it is critical in some treatments that the diameter ofthe Ox66™ particles should ideally be less than 300 nm as these particlesizes have less potential for toxicity and maximized efficacy

The blood additive compositions can include surface modifications ofnano-sized poly-oxygenated metal hydroxide particles with polyethyleneglycol (PEG) for increased vascular transit, protein for increasedsurface to volume ration, or specific charge to enhance absorption andsustained PO₂. These modifications of the poly-oxygenated metalhydroxide material as a blood additive extend the oxygenatingcapabilities of the material for longer periods of time, thus extendingproduct life, such as specifically in far-forward combat theatres.

This blood additive composition is extremely significant because theblood additive is agnostic to the blood type of a mammal, meaning thatthe blood additive can be administered to a human individual withouttyping the human individual's blood. Thus, even individuals with rareblood types can be effectively treated with the same blood additive.There is no time delay as the blood additive can be immediatelyadministered to an individual in a crisis situation. Further, the bloodadditive has significant shelf life and can be stored at roomtemperature in locations where administration of the blood additive canbe performed in emergency situations, such as in the battlefield toextend a soldier's life until the soldier can be transported to aquality hospital, or in an ambulance or fire truck. Stabilizing a humanindividual for hours or even minutes can save a human individual's life.

As shown in FIG. 1, this exemplary embodiment comprises a method 10 ofintravenously administering a mammal a therapeutic amount of acomposition including a poly-oxygenated metal hydroxide, such as a humanindividual, or an animal. The poly-oxygenated metal hydroxidecomposition may comprise a poly-oxygenated aluminum hydroxide, such asOx66™ particles. One method includes administration of a therapeuticallyeffective resuscitative fluid to increase tissue oxygenation PO₂ in themammal. Another method can include administration of a therapeuticallyeffective composition to treat a condition of a mammal. The methodcomprises preparing a mammal at step 12, such as preparing a site on themammal for receiving a catheter, and intravenously administering apoly-oxygenated metal hydroxide composition at step 14, such as using acatheter. Various methods and treatments are detailed as follows.

Study

A preclinical study was performed to ascertain the efficacy of apoly-oxygenated metal hydroxide in a mammal, comprising Ox66™ particles,and the details of the study and results are included. For this study,Particle Size A diameter is 100 um and Particle Size B diameter is 10um.

In this study, male Sprague-Dawley rats underwent a 50% blood volumeisovolemic hemodilution exchange with either Ox66™ or phosphate bufferedsaline (PBS; volume control), since Ox66M was suspended in PBS, such aslactated Ringers solution (LRS). LRS is a crystalloid electrolytesterile solution of specified amounts of calcium chloride, potassiumchloride, sodium chloride, and sodium lactate in water for injection.LRS is typically is used intravenously to replace electrolytes.Isovolemic hemodilution is the reduction of red blood cells (hematocrit)with an equal volume of hemodiluent, i.e., crystalloids, colloids oroxygen therapeutics.

The withdrawal/infusion rate was 2.0 ml×min⁻¹×kg⁻¹ and performed througha cannulated carotid artery and jugular vein. Systemic measurements wererecorded via a cannulated femoral artery that was connected to apressure transducer (MP150; Biopac Systems, Inc, Goleta, Calif.), whilemicrocirculatory parameters were collected through phosphorescencequenching and intravital microscopic examination of the exteriorizedspinotrapezius muscle. Compared to baseline, a 50% blood volume exchangewith either hemodiluent caused a reduction in heart rate, bloodpressure, arterial diameter and interstitial fluid (ISF) oxygen tension(PO₂) in all animals. However, Ox66™ animals demonstrated an improvementin ISF PO₂ compared to PBS animals. This finding demonstrates that Ox66™both transports and releases oxygen to the peripheral microcirculation.

Animals

-   -   Male Sprague Dawley rats (250-300 g)        Anesthetics    -   Isoflurane (induction)    -   Alfaxalone (continuous rate of infusion)

Surgical Preparation

-   -   Vessels and tracheal cannulation    -   Spinotrapezius muscle exteriorized

Systemic Parameters

-   -   BIOPAC MP150 (real-time analysis)

Tissue Oxygenation

-   -   Phosphorescence Quenching Microscopy        -   Palladium porphyrin (RO) probe distributed into            interstitium.        -   Phosphorescence decay curve captured and fit to standard            curve for translation to P_(ISF) O₂ in mmHg.

Hemodilution (HD)

-   -   Baseline parameters collected    -   50% isovolemic exchange of blood with test solution at 2.0        ml/kg/min    -   Post-HD parameters collected    -   Animals observed for 2 h post-HD

Hemodiluents

-   -   Phosphate Buffered Saline (PBS)    -   Ox66™ Size A [1×]    -   Ox66™ Size A [10×]    -   Ox66™ Size B [1×]    -   Ox66™ Size B [10×]

FIGS. 2A-2D show systemic characteristics of 50% isovolemic hemodilution(HID). Measurements were taken immediately prior to baseline (BL) andfollowing hemodilution at (HD t0). The volume exchange of whole bloodwith PBS (vehicle volume control) resulted in significant reductions inhematocrit, mean arterial pressure, and pulse pressure. The reduction inheart rate lacked statistical strength. ** p<0.01, *** p<0.001.

FIG. 3A shows tissue oxygenation (P_(ISF) O₂) following 50% volumereplacement. All P_(ISF) O₂ values (mmHg) were normalized to baseline(BL) for ease of comparison. PBS alone was used as a vehicle volumecontrol. Ox66™ compounds were suspended in PBS as crystalloids, whereparticle size A was 10× larger than particle size B and trended towardshigher oxygen delivery. Both particle sizes were assessed at 1× and 10×concentrations, but failed to show a concentration dependence of P_(ISF)O₂ in this range. * p<0.05 vs BL. Particle Size A diameter is 100 um andParticle Size B diameter is 10 um.

FIG. 3B shows tissue oxygenation (P_(ISF) O₂) following 50% volumereplacement. All P_(ISF) O₂ values (mmHg) were normalized to baseline(BL) for ease of comparison. PBS alone was used as a vehicle volumecontrol. FIG. 3B shows Ox66™ particles diameters being smaller thanthose shown in FIG. 3A that were suspended in PBS as crystalloids,having sizes of 300 nm, 1000 nm (1 um), 2500 nm (2.5 um), and 4800 nm(4.8 um), compared to the PBS alone. Compared to the results shown inFIG. 3A, Ox66™ particles having a diameter of around 10 urn suspended inPBS as a crystalloid appear to achieve a superior increase in P_(ISF) O₂values (mmHg).

FIG. 3B also shows Ox66™ particles suspended in a Colloid that resultsin vastly improved P_(ISF) O₂ values (mmHg) compared to PBS alone, andalso compared PBS including Ox66™ particles as a crystalloid havingreduced size particles, as shown. This is due in part to the bloodadditive composition including surface modifications of the nano-sizedpoly-oxygenated metal hydroxide particles with polyethylene glycol (PEG)for increased vascular transit, protein for increased surface to volumeration, and/or specific charge to enhance absorption and sustained PO₂.The PEGylation particles have a spherical shape that makes them moreslippery which results in better capillary transit and less irritationof the capillaries. The PEGylation also serves as an aggregateinhibitor. These modifications of the poly-oxygenated metal hydroxidematerial as a blood additive provides increased concentration controland extends the oxygenating capabilities of the material for longerperiods of time, thus extending product life, such as specifically infar-forward combat theatres,

FIG. 3C shows the results of resuscitation of five male Sprague-Dawleyrat specimens after hemorrhagic shock. As shown, two specimens underwentresuscitation with a Colloid including 2.4 um Ox66™ PEGylationparticles, and each specimen survived 1 hour after hemorrhagic shock.This is significant as death would have occurred within 10 minutes ofhemorrhagic shock.

Even more significant, one of the three specimens that underwentresuscitation with a Colloid including 4.8 um Ox66™ PEGylation particlesshowed a significant immediate increase in P_(ISF) O₂, and survived 8hours after hemorrhagic shock, when the monitoring was completed and thespecimen continued to survive, a complete survival. A second specimenshowed a significant immediate increase in P_(ISF) O₂ and survived 3hours. The third specimen also survived an additional 3 hours. Thissignificant survival of all five specimens after hemorrhagic shock byresuscitating each with a Colloid including Ox66™ PEGylation particlesis remarkable. Advantageously, survival from hemorrhagic shock withoutusing a blood product is extremely encouraging, as the Colloid does notrequire blood typing. When used on individuals on the battlefield, thissurvival time is significant and allows transport of an individual thatundergoes hemorrhagic shock to a treatment facility.

FIGS. 4A and 4B shows systemic parameters following isovolemichemodilution with test solutions. HD=Hemodilution; tn=time point inminutes following hemodilution. As shown in FIG. 4A, heart ratesgenerally followed the scheme of slowing down by HD t0 and thenreturning to baseline by t60. As shown in FIG. 4B, mean arterialpressure remained low, but stable following hemodilution with theexception of Size A at 10× concentration. Statistical tests were notperformed due to low sample sizes (N=2-4).

FIG. 5 shows arteriolar luminal diameters. Arterioles included weresmaller than 60 microns at baseline.

SUMMARY

The ‘50% Isovolemic Hemodilution’ model produces a good reduction insystemic cardiovascular parameters and tissue oxygenation to assesstherapeutic potential of interventions.

Ox66™ is capable of carrying and delivering oxygen to hypoxic peripheraltissues.

Administering Surface Modified Ox66™ Particles

In an exemplary embodiment, the administered Ox66™ particles may besurface modified for specific therapeutic uses such as time release,PEGylation, growth factor modification, antibacterial, antimicrobial,protein modification, and enzymes.

Treatment of Traumatic Brain Injury (TBI), Strokes, and CTE

To achieve microcirculation in mammals, such as to treat TBI andstrokes, the Ox66™ particles preferably have a diameter of less than 300nm to pass the blood brain barrier (BBB). The upper limit of pore sizeenabling passive flow across the BBB is usually <300 nm; however,particles having a diameter of several nanometers can also cross the BBBvia carrier-mediated transport using specialized transport proteins.Alternatively, receptor-mediated transport can act as an “escort” forlarger particles. This exemplary embodiment comprising intravenouslyadministering a therapeutic amount of a composition including Ox66™particles having a diameter of less than 300 nm is therapeuticallyeffective in treating a mammal with TBI and BBB. This is anextraordinary accomplishment, and can revolutionize the treatment of notonly TBI and BBB, but also other brain conditions/injury includingChronic Traumatic Encephalopathy (CTE), which is a progressivedegenerative disease of the brain found in athletes, military veterans,and others with a history of repetitive brain trauma.

Treatment of Diabetes

To achieve microcirculation in mammals to treat Diabetes, this exemplaryembodiment comprises intravenously administering to a mammal atherapeutic amount of a composition including Ox66™ particles as a fluidthat is therapeutically effective to increase PO₂ in the mammal, such asa human individual, or an animal, to reduce the effects of Diabetes.

Treatment of Carcinoma

To treat cancer in mammals, exemplary embodiments comprise intravenouslyadministering to a mammal a therapeutic amount of a compositionincluding Ox66™ particles as a fluid that is therapeutically effectiveto reduce the effects of, or eliminate, cancer cells in the mammal, suchas a human individual, or an animal. The composition Ox66™ can also beadministered orally to the mammal.

The charts in the Figures described hereafter illustrate laboratoryresults of the proliferation of the identified carcinoma afteradministration of various concentrations of the Ox66™ in a fluid toliving carcinoma cells compared to control, which is no administrationof the Ox66™ to the cells.

For the following results, three assays are used: Janus Green (JG)colorimetric assay, Lactase Dehydrogenase (LDH) colorimetric assay, andCFDA-5 fluorometric assay.

Janus Green (JG) is a supravital stain, meaning it is absorbed bydamaged cells. It is not able to penetrate healthy cells, but when cellsare damaged or dead, it is able to pass easily into the cell, and stainthe mitochondria. Janus Green is a relatively quick way to assess theheath of cells, and it must be used in two parts; one plate forviability, and the other for proliferation in order to obtain a percentviability of cells. The measurements are not exact numbers, but ratheran estimate based on professional observation.

Janus Green Protocol:

Obtain two (2) 96-well plates (one plate for viability, the other platefor proliferation). Seed ˜1 Million identified living carcinoma cellsper plate.

Once the carcinoma cells have reached 50% confluency (˜24 hours), dosethe cells in the plates with varying concentrations of Ox66™ fluid (2columns of cells for each concentration of Ox66™ including control).

After 24 hours, run JG.

Standard Protocol was followed:

For the viability, the cells were stained with JG dye before being fixedwith 100% ethanol. This shows which cells were still alive.

For the proliferation, the cells were fixed with 100% ethanol beforebeing stained with JG to get an approximate number of how many cellswere seeded.

The plates were then run in a colorimetric plate reader.

Lactate dehydrogenase is an enzyme that is present in all living cells,and is released when cell membrane integrity is compromised, making thisassay, which detects the presence of LDH a reliable option forcytotoxicity. The LDH assay uses the compound iodonitrotetrazolium (INT)to react with LDH present to form a red colored formazan. This react canthen be read under a colorimetric plate reader and be quantified.

LDH Protocol:

Seed and dose the carcinoma cells the same as for JG, with only one96-well plate.

50 microliters of cell media are taken from each well and placed into anew well plate, then 50 microliters of LDH solution is added to the newwell plate, along with the media.

The plate was then run in a colorimetric plate reader.

5-CFDA, AM assay is an enzymatic marker assay, as well as a cellmembrane permeability marker. Enzymatic activity present within thecells will cause the CFDA dye to fluoresce, and the cell membraneintegrity will retain the fluoresced product within the cell.

5-CFDA, AM Protocol:

Seed and dose the cells the same as for LDH.

The cells are stained with the CFDA dye and are incubated for ˜30minutes, then the solution is diluted with media, and read under afluorescent plate reader.

Study 1—Liver Carcinoma (HEPG-2)

The proliferation of hepatocarcinoma cells (HEPG-2) was significantlyreduced following administration of various concentrations of Ox66™ tothe cells, as shown in FIG. 6. A hypoxic microenvironment, which is acommon feature of hepatocellular carcinoma can induce HIF-1α expressionand promote the epithelial-mesenchymal transition (EMT). Additionally,it can induce the invasion of cancer cells. This proven characteristicof hepatocarcinoma supports the hypothesis that Ox66™ is effective inreducing the proliferation of these cells.

Images shown in FIG. 7A and FIG. 7B illustrate HEPG-2 cells prior todosing and after dosing with Ox66™ fluid, respectively.

Study 2—Prostate Carcinoma (22Rv1)

The proliferation of prostrate carcinoma (22Rv1) cells was significantlyreduced following administration with various concentrations of Ox66™fluid to the cells, as shown in FIG. 8. Prostrate carcinoma cells arehypoxic, which helps explain why Ox66™ is effective in reducing theproliferation of these cells.

For this cell line, 22Rv1 (prostate carcinoma), the Janus Greencolorimetric assay was used to determine how viable the cells were afterbeing dosed with varying concentrations of the Ox66 ™ into the cellculture media. This administration is similar to injection into theblood stream as would be given via an intravenous injection (IV). JanusGreen is an exclusion dye, which only stains mitochondria and nuclei ofdamaged cells. For the assay, the cell culture was washed twice withphosphate buffered saline (PBS), followed by one minute fixation withabsolute ethanol. The culture was then subjected to one-minute stainingby Janus Green B dye solution followed by two PBS wash to remove theexcess dye. Then the encapsulated dye from these cells was extractedwith absolute ethanol, and an additional 100 ul water was added to eachwell to maintain samples. Optical intensity was then read at 630 nm on amicroplate reader. Janus Green gives intensive staining of the nucleiwith light staining of the cytoplasm, thus outlining cells clearly.Therefore, morphologic changes of cells can also be screened after theassay using an inverted microscope. The more Janus Green present, themore damaged or dead cells are present as well. The graph shows that foradministration of Ox66™ fluid to the cells at a concentration of 100mg/L, there is a statistical difference between the uptake of JanusGreen at 100 mg/L than at 0 mg/L, or the control. This is the onlyconcentration that is statistically different when compared to thecontrol for this carcinoma.

Study 3—Lung Carcinoma (A549)

The proliferation of lung carcinoma (A549) cells was significantlyreduced following administration with various concentrations of Ox66™fluid to the cells, as shown in FIG. 9. Lung carcinoma cells arehypoxic, which helps explain why Ox66™ is effective in reducing theproliferation of these cells.

For this cell line, A549 (lung carcinoma), the Janus Green colorimetricassay was used to determine how viable the cells were after being dosedwith varying concentrations of Ox66™ into the cell culture media. Thisadministration is similar to injection into the blood stream as would begiven via an intravenous injection (IV). Janus Green is an exclusiondye, which only stains mitochondria and nuclei of damaged cells. For theassay, the cell culture was washed twice with phosphate buffered saline(PBS), followed by one minute fixation with absolute ethanol. Theculture was then subjected to one-minute staining by Janus Green B dyesolution followed by two PBS wash to remove the excess dye. Then theencapsulated dye from these cells was extracted with absolute ethanol,and an additional 100 ul water was added to each well to maintainsamples. Optical intensity was then read at 630 nm on a microplatereader. Janus Green gives intensive staining of the nuclei with lightstaining of the cytoplasm, thus outlining cells clearly. Therefore,morphologic changes of cells can also be screened after the assay usingan inverted microscope. The more Janus Green present, the more damagedor dead cells are present as well. The graph shows that for theadministration of Ox66™ at 50 mg/L and 100 mg/L there is a statisticaldifference between the uptake of Janus Green at 50 mg/L and 100 mg/Lthan at 0 mg/L, or the control. This indicates that these carcinomacells are more receptive to the Ox66™ treatment than 22Rv1 cells.

Study 4—Colon Adenocarcinoma (CaCo-2)

The proliferation of colon adenocarcinoma cells (CaCo-2) wassignificantly reduced following administration with variousconcentrations of Ox66™ in the culture media of the cells, as shown inFIG. 10. Colon adenocarcinoma cells are hypoxic, which helps explain whyOx66™ is effective in reducing the proliferation of these cells.

For this cell line, CaCo-2 (colon adenocarcinoma), the Janus Greencolorimetric assay was used to determine how viable the cells were afterbeing dosed with varying concentrations of Ox66™ into the cell culturemedia. This administration is similar to injection into the blood streamas would be given via an intravenous injection (IV). fluid. Janus Greenis an exclusion dye, which only stains mitochondria and nuclei ofdamaged cells. For the assay, the cell culture was washed twice withphosphate buffered saline (PBS), followed by one minute fixation withabsolute ethanol. The culture was then subjected to one-minute stainingby Janus Green B dye solution followed by two PBS wash to remove theexcess dye. Then the encapsulated dye from these cells was extractedwith absolute ethanol, and an additional 100 ul water was added to eachwell to maintain samples. Optical intensity was then read at 630 nm on amicroplate reader. Janus Green gives intensive staining of the nucleiwith light staining of the cytoplasm, thus outlining cells clearly.Therefore, morphologic changes of cells can also be screened after theassay using an inverted microscope. The more Janus Green present, themore damaged or dead cells are present as well. The graph shows that foradministration of Ox66™ at 50 mg/L and 100 mg/L there is a statisticaldifference between the uptake of Janus Green at 50 mg/L and 100 mg/Lthan at 0 mg/L, or the control. This indicates that these cells are morereceptive to Ox66™ than 22Rv1 cells. There is a substantial jump inuptake of the Janus Green at 100 mg/L, meaning there were many moredamaged cells at this concentration.

Erectile Dysfunction

To achieve the treatment of erectile dysfunction in mammals, thisexemplary embodiment comprises intravenously administering to a mammal atherapeutic amount of a composition including Ox66™ particles that istherapeutically effective to increase oxygenated blood flow thusmitigating physical dysfunction in the mammal, such as a humanindividual, or an animal, to reduce the effects of erectile dysfunction.In another embodiment, the Ox66™ particles could be embodied in a tabletor capsule form and administered orally.

Sickle Cell Anemia

To achieve the treatment of sickle cell anemia in mammals, thisexemplary embodiment comprises intravenously administering to a mammal atherapeutic amount of a composition including Ox66™ particles (˜0.07 μm)that is therapeutically effective to increase oxygenated blood flow thusmitigating dysfunction in the mammal, such as a human individual, or ananimal, to reduce the effects of sickle cell anemia. In anotherembodiment, the Ox66™ particles could be embodied in a tablet or capsuleform and administered orally. In sickle cell anemia, the red blood cellsbecome rigid and tacky and are shaped like sickles hence the name of thedisease. These irregularly shaped “sickle” cells do not move throughsmall blood vessels, resulting in slowing or blockage of blood flow andoxygen to parts of the body. This embodiment of Ox66M particles couldoxygenate the body in a crisis and act as an alleviation strategy forsickle cell anemia.

Bronchopulmonary Dysplasia (BPD)

To treat bronchopulmonary dysplasia in mammals, this exemplaryembodiment comprises intravenously administering to a mammal atherapeutic amount of a composition including Ox66™ particles as a fluidthat is therapeutically effective to reduce the effects of, oreliminate, BPD in the mammal, such as a human individual, or an animal.A critical problem facing preterm infants is adequate lung function.Premature babies can have strokes, chronic lung disease and potentialbrain damage due to small, fragile blood vessels, and pressurized oxygenrequired after birth to keep the lungs functional. There is a need foran alternative oxygen therapy that mitigates the aforementioned risks.These preemies frequently encounter complications such as chronic lungdisease—sometimes called bronchopulmonary dysplasia (BPD). BPD can occurbecause the infants still have some inflammation in their lungs and mayrequire extra oxygen or medications to help them breathe comfortably.There are several hyper-oxygenated associated illnesses that a preterminfant will suffer such as retinopathy of prematurity (ROP),periventricular leukomalacia, cerebral palsy, and the previouslymentioned bronchopulmonary dysplasia (BPD), to name a few.Administration of Ox66™ provides alternative oxygen delivered by lessinvasive means yet supplying oxygen to the preterm infant.

Alzheimer's Disease (AD)

To treat Alzheimer's disease in mammals, this exemplary embodimentcomprises intravenously administering to a mammal a therapeutic amountof a composition including Ox66™ particles as a fluid that istherapeutically effective to reduce the effects of, or eliminate, AD inthe mammal, such as a human individual, or an animal. Alzheimer'sdisease (AD) is classified as a neurodegenerative disorder. The causeand progression of the disease are not well understood. AD is associatedwith hallmarks of plaques and tangles in the brain. Current treatmentsonly help with the symptoms of the disease and there are no availabletreatments that stop or reverse the progression of the disease. As of2012, more than 1,000 clinical trials have been or are being conductedto test various compounds in AD. There is currently no approved drugtherapy for AD that will stop or reverse the progression of the disease.There is a clear link between low oxygen levels in the brain andAlzheimer's disease, but the exact mechanisms behind this are not yetfully understood (Alzheimer's Society, Proceedings of the NationalAcademy of Sciences). A healthy brain needs a good supply of oxygen. Adisruption of the blood flow through or to the brain causes low oxygenlevels. When there is damage or a blockage, or the blood supply itselfis low in oxygen then insufficient oxygen will be delivered to the braincells. Ox66™ offers the potential of micrometer sized (˜0.07 μm)particles increasing oxygen delivery to the brain. With this offloadingof oxygen, there is significant potential to mitigate the developmentand/or the progression of AD.

Autism

To treat autism in mammals, this exemplary embodiment comprisesintravenously administering to a mammal a therapeutic amount of acomposition including Ox66™ particles as a fluid that is therapeuticallyeffective to reduce the effects of, or eliminate, autism in the mammal,

What is claimed is:
 1. A method of treating a mammal, comprisingadministering a therapeutically effective amount of a poly-oxygenatedaluminum hydroxide composition to the mammal in need thereof to reduce aproliferation of colon adenocarcinoma CaCo-2 cells, wherein thepoly-oxygenated aluminum hydroxide composition comprises a clathratecontaining fee oxygen gas (O2) molecules; and wherein thetherapeutically effective amount of the poly-oxygenated aluminumhydroxide composition ranges from 10 mg/L to 1000 mg/L.
 2. The method asspecified in claim 1 wherein the poly-oxygenated aluminum hydroxidematerial is configured to provide bioavailable oxygen gas molecule tothe mammal when administered to the mammal.
 3. The method as specifiedin claim 1 wherein the poly-oxygenated aluminum hydroxide composition isadministered intravenously.
 4. The method as specified in claim 1wherein the poly-oxygenated aluminum hydroxide composition isadministered direct to the colon adenocarcinoma CaCo-2 cells.
 5. Themethod as specified in claim 1 where the poly-oxygenated aluminumhydroxide composition is administered orally.
 6. The method as specifiedin claim 1 wherein the compsition comprises a fluid.
 7. The method asspecified in claim 1 wherein the poly-oxygenated aluminum hydroxidecomposition soluble in the fluid.
 8. The method as specified claim 6wherein the fluid is a phosphate buffered saline (PBS).
 9. The method asspecified in claim 6 wherein the fluid is lactated ringers solution(LRS).
 10. The method as specified in claim 1 wherein thepoly-oxygenated aluminum hydroxide composition comprises poly-oxygenatedaluminum hydroxide particles all having a diameter of less than or equalto 10 um.
 11. The method as specified in claim 10 wherein thepolyoxygenated aluminum particles are homogeneous.
 12. The method asspecified in claim 1 wherein the poly-oxygenated aluminum hydroxidecomposition comprises poly-oxygenated aluminum hydroxide particles allhaving a diameter of less than or equal to 1 um.
 13. The method asspecified in claim 1 wherein the poly-oxygenated aluminum hydroxidecomposition comprises poly-oxygenated aluminum hydroxide particles thatare surface modified.
 14. The method as specified claim 13 wherein thepoly-oxygenated aluminum hydroxide material comprises particles that aresurface modified with polyethylene glycol (PEG).
 15. The method asspecified in claim 1 wherein the mammal is a human individual.